Single-Cell Western Blotting to Determine Cell Heterogeneity

1. Determining Cell Composition in the Cell Suspension Using Single-cell Western Blot (scWB) Analysis

1. Rehydrate the scWB chip before use in 15 mL of 1x suspension buffer in a Petri dish for at least 10 min at RT.
2. Add 1 mL of the diluted single-cell suspension (10,000-100,000 cells/mL) to the rehydrated chip. Set the surface onto the bottom of a 10 cm Petri dish. Ensure that the surface is set flat.
3. Cover the Petri dish with a lid to prevent drying and let the cells settle for 5-15 min by a gradient.
4. Place the chip under a bright-field microscope and inspect the wells at 10x magnification.
   NOTE: Approximately 15-20% of microwells should be occupied by a single cell, and fewer than 2% of wells should contain 2 or more cells.
5. Tilt the 10 cm Petri dish containing the chip by 45 degrees.
6. Wash the chip with the 1x suspension buffer to remove uncaptured cells from the surface of the chip by gentle pipetting from the top to the bottom of the chip. Repeat 3 times.
7. Prepare the single-cell Western instrument.
8. Set the lysis time, electrophoresis time, and UV capture time. To analyze recruited macrophages recovered from the basement membrane-derived gel plug, use the following settings: lysis time: 0 s; electrophoresis time: 160 s; UV capture time 240 s.
9. Carefully load the chip into the electrophoresis cell of single cell Western instrument, gel-side facing. Use caution not to damage the gel side of the chip.
10. Pour the lysis/running buffer into the chamber and completely cover the entire single-cell Western chip. Start the cell lysis.
11. Run the scWB instrument using the setting listed in 1.8.
12. Once the run is complete, transfer the chip to a 10 cm Petri dish and wash the chip twice with 1x washing buffer for 10 min at RT.
13. Prepare dilutions of primary antibody (anti-vinculin: 1:10; anti-CD45: 1:15, anti-CD11b: 1:20, anti-F4/80: 1:10) in a total volume of 80 µL of antibody diluent (mix 8 µL of antibody 1 plus 8 µL of antibody 2 plus 64 µL of diluent).
14. Add 80 µL of the primary antibody solution to the antibody probing chamber and lower the chip gel-side down so that the antibody solution wicks across the chip.
15. Incubate with the primary antibody solution for 2 h at RT.
16. Wash the chip three times for 10 min in 1x washing buffer on the shaker.
17. Wash the chip once for 10 min in the water on the shaker to desalt the gel.
18. Prepare a 1:20 dilution of secondary antibody in a total volume of 80 µL (mix 2 µL of secondary antibody plus 78 µL of diluent).
19. Incubate the chip with a secondary antibody for 1 h at RT protected from light.
20. Wash the chip three times for 10 min with 1x washing buffer on the shaker.
21. Spin the chip using a slide spinner to remove any remaining washing buffer.
22. Scan the chip on a dual-laser microarray scanner at 5 µm resolution at the spectral channel of the fluorophore coupled to the secondary antibodies.
23. Analyze data using scWB-specific software.

2. Stripping the scWB Chip

1. Store the scanned chip in a wash buffer until ready to strip.
2. Place the water bath in a fume hood.
3. Place a 50 mL tube rack in the water bath with the water just 1 cm above the rack and set the water temperature at 60 °C.
4. Prepare the stripping buffer. For 1 L of stripping buffer solution, dissolve 9.85 g of Tris-HCl (pH 6.8) and 20 g of SDS in 900 mL of distilled water and adjust pH. Then fill with distilled water to 1L. Add 0.8% (v/v) of β-mercaptoethanol (β-ME; 14.3 M) immediately before each use.
5. After the initial scan, place the chip in a 10 cm Petri dish before stripping.
6. For each chip, take 40 mL of stripping buffer and add 320 µl of β-ME.
   CAUTION: β-ME is toxic and hazardous to humans and the environment. The following procedure should be done in a fume hood.
7. Place the chip in the canister and seal the canister with parafilm to prevent water from leaking into the canister
8. Place the canister inside the tube rack in the pre-warmed water bath.
9. Incubate for 90 min.
10. Carefully remove the chip from the canister and place it in a fresh Petri dish.
11. Briefly wash the chip once with 1x wash buffer. Then add 15 mL of 1x washing buffer to the Petri dish.
12. Wash the chip for 15 min on a shaker. Repeat the wash step four times.
    NOTE: Chip is ready for the next primary antibody (see steps 1.12 – 1.21)